1. Field of the Invention
This invention relates to a method for receiving and storing biological molecules, and more specifically, this invention relates to a method for storing proteins in their native state for assay or application and/or delivery to sites remote from the initial extraction and storage facilities.
2. Background of the Invention
Membrane proteins represent an extremely important class of biomolecules whose functions are vital to human health. As such, they comprise the vast majority of drug targets being pursued at present. In contrast to soluble proteins, much less is known about membrane proteins, primarily due to the difficulty of producing sizeable quantities of them in natively-folded, functional, and relatively pure form.
In order for membrane proteins to subsist in the aqueous environments required for most chromatographies and analyses, they must first be removed from their native lipid bilayer, usually through the use of detergents, and remain soluble in detergent environments for further characterization or subsequent crystallization attempts. Membrane protein-detergent complexes, upon purification, are most frequently preserved either through flash freezing, the use of non-native, synthetic architectures, or by reconstitution of samples into liposomes.
Flash freezing is often employed in attempts to extend the viability and structure of some biomolecules. Indeed, it is well established that the function of most membrane proteins is short-lived, sometimes just hours, when removed from their native environs. Although flash freezing works successfully for some protein and detergent combinations, it is hardly a universal tool for extending the viability of proteins in their native states or maintaining the native structure of the proteins. Generally, cycling across the liquid-solid transition (i.e., the action of freezing and thawing) has deleterious effects on the integrity of samples and excludes the use of this approach, irrespective of the length of storage in the frozen state.
Non-native, synthetic architectures have been explored as possible biomolecule preservation systems. The “maintenance” and sequestration of membrane proteins in non-native, synthetic architectures that mimic native membranes have involved the use of various model systems that can be grouped into three general types: monolayer, planar bilayer, or mesophases. However, each of these systems have their drawbacks.
The study of monolayers at an air-water interface via the use of a Langmuir trough is well suited for measurement of surface activity, but it models only the outer leaflet of the bilayer and, thus, does not provide an appropriate model for the study of molecular species that fully insert into a lipid bilayer. Similarly, although supported lipid bilayers provide a more accurate model of natural biomembranes, they suffer from a serious limitation in that the underlying substrate (typically, glass) can interact detrimentally with molecules that fully insert into the bilayer (e.g., not providing a suitable water layer).
Bicelles (oblate bilayer micelles) offer an improvement over simple micelles since their architecture better models a native biomembrane. Wide-scale implementation of bicelles is limited, however, since stable field alignment can be induced only over a narrow, often elevated temperature range. In addition, undesirable bicelle surface-protein associations can lead to phase separation and poor temporal stability. A. T. Brunger et al. Acta Crystallographica D54, 905 (1998).
Phospholipid bilayer discs serving as model membranes also have been utilized to store proteins. These nanodiscs consist of a membrane scaffold protein and a detergent solubilized phospholipid, and self-assemble upon addition of the desired protein and removal of the detergent used in the isolation and purification of the membrane protein target. The protein-stabilized assemblies, however, tend to be heterogeneous, complicate certain spectroscopic measurements, and inhibit interactions among protein partners that are frequently required for proper function.
Liposomes are aqueous compartments enclosed by lipid membranes. Liposomes represent a better alternative to freezing in many cases. Micelles and liposomes have been successfully employed in fundamental physicochemical studies (liposomes have primarily been used in functional assays with considerably less in the area of structure) and have found some practical application in the area of drug delivery and DNA transfection. D. D. Lasic, D. Needham, Chem. Rev. 95, 2601 (1995). C. R. Safinya, Curr Opin Struct Biol 11, 440 (August, 2001). However, because they do not possess long-range translational order (diffraction peaks) and cannot be ordered asymmetrically, they are not candidate materials from which detailed structural information of guest membrane proteins can be obtained. Membrane proteins reconstituted into liposomes are not amenable to many assays commonly employed to probe structure and function. Furthermore, to remove membrane proteins from liposomes, one must re-isolate protein components by reintroducing a high concentration of detergent into the sample.
A need exists for a method to preserve proteins in their native state to make further characterization of their structures and functions possible. The method should facilitate indefinite storage of the protein and, where possible, allow in situ characterization of the native protein to assess its structural integrity and functional state.
A need also exists for a biocompatible medium capable of providing a cellular mimetic environment for the encapsulation and organization of a wide range of proteins (including soluble and membrane) and complexes. Ideally, such a medium should possess the ability to be highly ordered. In this way, it could be adapted to low-to-medium resolution structural studies of the organized proteins using a range of techniques, including NMR and EPR spectroscopies, small-angle X-ray or neutron scattering/diffraction, and optical spectroscopy.